Laser scanning microscopies (LSM) include some of the most important imaging modalities in biology. For example, confocal laser scanning microscopy (CLSM) is the tool of choice for high-resolution fluorescence microscopy in cultured and fixed tissue preparations [1
]. Two-photon excitation laser scanning microscopy (2PLSM) is ideal for fluorescence microscopy in scattering media, such as living skin and brain tissue [4
Historically, new developments and applications of laser scanning microscopy have been limited primarily by the availability of new hardware. The development of suitable laser light sources and powerful data acquisition systems running on desktop computers brought CLSM to fruition [3
]. In the case of 2PLSM, the development of mode-locked pulsed light sources operating in the red and near IR spectral range [6
] made it practical [5
Several lines of commercial CLSMs and 2PLSMs are available. A manufacturer typically will offer one principal model and allow the customer to choose between different versions and options. Fundamentally, in terms of optics and software, a single general purpose design is offered. However, exciting applications demand specialized design. Often these applications include some of the most promising experimental developments. Currently, most hardware components of CLSMs and 2PLSMs (lasers, optics, scanning mirrors, detectors, and computers) are mature and readily available. The development of cutting edge applications using custom design is often limited by the availability of suitable software to control laser scanning microscopes.
In our laboratory we have written flexible software to control LSMs (ScanImage
). In a LSM, a signal (typically fluorescence) is detected as a focused laser beam is scanned across a sample. The signal emitted from within a number of tiny, contiguous regions (called pixels) [3
] is collected over short time intervals (the pixel time, Tp
). The integrated signal is converted to a digital pixel value and the collection of all the pixels from the scanned region defines the image. The location of the pixel in the image is determined by the location of the laser at the time of acquisition. Synchronization of the position of the laser beam and the signal collection is therefore essential for imaging.
In order to time scanning and acquisition precisely and to achieve high performance in terms of signal-to-noise ratio, on-line averaging, and real-time image refresh, many commercially available laser-scanning microscopes (e.g. Olympus Fluoview™, Olympus America Inc., Melville, NY; Bio-Rad Radiance 2100™, Bio-Rad Laboratories, Hercules, CA) use proprietary signal conditioning electronics and data acquisition boards. Commercial systems are suitable for many applications and have also been customized for specific experimental needs [4
]. However, complete custom design is often more advantageous and cost effective [11
]. Two major impediments to custom design are the specialized software and hardware required for laser scanning microscopy.
Here we present a software package called ScanImage
for collection of laser-scanned images that functions at a high level without the need for elaborate custom hardware [14
]. The key idea is to use fast data acquisition boards and CPU-based numeric computations to perform most of the tasks that are accomplished by DSP boards and analog integrators in typical commercial systems. Thus ScanImage
simplifies the design and construction of custom microscopes. An additional advantage is that ScanImage
is written in MATLAB (version 6.1 or later; MathWorks Inc., Natick, MA), and thus already fully integrated into a high-level image analysis environment.